Methods and compositions for generating asymmetrically-tagged nucleic acid fragments

ABSTRACT

The present disclosure provides improved methods for generating asymmetrically-tagged nucleic acid constructs, compositions comprising such constructs, and kits and systems for generating such constructs.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation application of U.S. patentapplication Ser. No. 15/617,294, filed Jun. 8, 2017, which claims thebenefit of priority to U.S. Provisional Patent Application 62/351,500,filed Jun. 17, 2016, which is hereby incorporated by reference herein inits entirety.

BACKGROUND OF THE INVENTION

The ability to understand the genetic code that serves as the blueprintfor the framework of all life has yielded countless advances incountless areas. From the ability to diagnose disease to the ability toidentify evolutionary connections and/or diversity, to the ability tomanipulate the genetic framework in the development of new materials andcompositions, this understanding has opened doors to innumerableadvances that have benefitted and will continue to benefit the humanrace.

Integral to these advances have been the advances in technology directedto the reading and/or characterization of the genetic code. For example,development of nucleic acid sequencing technologies has allowed for thebase-by-base identification of the nucleic acid sequences that make upthe genetic code to the point that entire human genomes have beenelucidated. Other advances include rapid array-based technologies thatallow reasonably facile identification of genetic patterns from patientsor other biological samples.

With each technological advance, there exist opportunities to furtherimprove the state of the art through advances in related or ancillarytechnologies associated with those advanced areas. For example, advancesin fluorescent dye chemistries have fueled many advances in genetictechnologies by permitting simple optical analyses of biologicalreactions and their products. Likewise, development of microfluidictechnologies has provided for advances in fluid and reagent handling toyield a reproducibility that had not been previously achievable throughmore conventional means.

The present disclosure is directed to improved processes, systems andcompositions for generating asymmetrically-tagged nucleic acids thatfind use in a number of different downstream genetic analyses.

BRIEF SUMMARY OF THE INVENTION

The present disclosure provides improved methods for generatingasymmetrically tagged nucleic acid constructs, compositions comprisingsuch constructs, and kits and systems for generating such constructs.

Aspects of the present disclosure include methods of producing anasymmetrically-tagged nucleic acid, comprising: providing adouble-stranded nucleic acid fragment, wherein the 5′ ends of thenucleic acid fragment are dephosphorylated; ligating a hairpin adapterto the double-stranded nucleic acid fragment to produce a first ligationproduct, wherein the first ligation product comprises nicks at thedephosphorylated 5′ ends of the nucleic acid fragment; contacting thefirst ligation product to a polymerase, wherein the polymerase binds tothe nicks in the first ligation product; incubating thepolymerase-contacted first ligation product under nucleic acid synthesisconditions, wherein the polymerase extends the adapter in atemplate-dependent nucleic acid synthesis reaction to produce a nucleicacid synthesis product; ligating a second adapter to the nucleic acidsynthesis product, wherein the second adapter is ligated to the endopposite the hairpin adapter on the nucleic acid synthesis product, andwherein the second adapter has at least one difference as compared tothe hairpin adapter, thereby producing an asymmetrically-tagged nucleicacid.

In certain embodiments, the providing step comprises fragmenting anucleic acid sample to generate the double-stranded nucleic acidfragments.

In certain embodiments, the providing step further comprisesdephosphorylating the 5′ ends of the double-stranded nucleic acidfragments.

In certain embodiments, the second adapter is a second hairpin adapter.

In certain embodiments, the second adapter is selected from the groupconsisting of: a bubble adapter, double-stranded linear adapter, alinear overhang adapter, and a Y adapter.

In certain embodiments, the contacting step is performed prior to andindependently from the incubating step.

In certain embodiments, the polymerase is a strand displacing polymeraseenzyme.

In certain embodiments, the strand displacing polymerase is selectedfrom the group consisting of: a Φ29 DNA polymerase or modified versionthereof, a homolog of a Φ29 DNA polymerase or modified version thereof,and combinations thereof.

In certain embodiments, the method further comprises contacting thenucleic acid synthesis product with a kinase under conditions forphosphorylating the free 5′ end of the nucleic acid synthesis product.

In certain embodiments, the method further comprises determining anucleotide sequence of at least one strand of the asymmetrically-taggednucleic acid using a template directed, polymerase mediated nucleic acidsequencing process.

In certain embodiments, the sequencing process sequences each strand ofthe double stranded region of the asymmetrically-tagged nucleic acid.

In certain embodiments, the sequencing process sequences at least onestrand of the double stranded nucleic acid segment more than once.

In certain embodiments, at least one of the first hairpinoligonucleotide and the second hairpin oligonucleotide comprises aprimer recognition sequence.

In certain embodiments, at least one of the first and second hairpinoligonucleotides comprises a barcode sequence.

Aspects of the present disclosure include methods of sequencing anucleic acid, comprising: producing an asymmetrically-tagged templatenucleic acid comprising a first adapter, an insert, and a second adapteras set forth above; and monitoring nucleotides incorporated in atemplate dependent synthesis reaction to identify a sequence ofnucleotides in the insert of the asymmetrically-tagged template nucleicacid.

Aspects of the present disclosure include methods of sequencing nucleicacids from a plurality of samples, comprising: preparingasymmetrically-tagged template nucleic acid segments from each of aplurality of discrete nucleic acid samples as set forth above (as in anyone of originally filed claims 1 to 14), wherein theasymmetrically-tagged template nucleic acid segments comprise asample-specific barcode sequence in at least one adapter sequence;pooling the asymmetrically-tagged template nucleic acid segments fromthe plurality of discrete nucleic acid samples; sequencing the templatenucleic acid segments pooled in the pooling step; and identifyingnucleic acid sequences as deriving from a discrete nucleic acid samplebased at least in part on the barcode sequence identified in thesequencing step.

Aspects of the present disclosure include template preparation kitscomprising: a first hairpin adapter; a second adapter, wherein thesecond adapter comprises at least in difference from the first hairpinadapter; a phosphatase; a kinase; a nucleotide polymerase; a ligase; andone or more buffers or reagents for performing de-phosphorylationreactions, ligation reactions, phosphorylation reactions, and nucleicacid synthesis reactions on a double stranded nucleic acid fragment.

In certain embodiments, the kit further comprises an exonuclease.

In certain embodiments, the polymerase is a strand displacing polymeraseenzyme.

In certain embodiments, the strand displacing polymerase is selectedfrom the group consisting of: a Φ29 DNA polymerase or modified versionthereof, a homolog of a Φ29 DNA polymerase or modified version thereof,and combinations thereof.

In certain embodiments, the kit further comprises a component fornucleic acid isolation, nucleic acid enrichment, and/or nucleic acidsize selection.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a first embodiment of generatingasymmetrically-tagged nucleic acid fragments.

FIG. 2 illustrates the nucleic acid synthesis product from analternative first ligation product.

FIG. 3 illustrates the nucleic acid synthesis product from anotheralternative first ligation product.

FIG. 4 illustrates alternative second adapter structures and theresulting asymmetrically-tagged target nuclei acids.

DETAILED DESCRIPTION OF THE INVENTION

The practice of the present invention may employ, unless otherwiseindicated, conventional techniques and descriptions of organicchemistry, polymer technology, molecular biology (including recombinanttechniques), cell biology, biochemistry, and immunology, which arewithin the skill of the art. Such conventional techniques includepolymer array synthesis, hybridization, ligation, phage display, anddetection of hybridization using a label. Specific illustrations ofsuitable techniques can be had by reference to the example herein below.However, other equivalent conventional procedures can, of course, alsobe used. Such conventional techniques and descriptions can be found instandard laboratory manuals such as Genome Analysis: A Laboratory ManualSeries (Vols. I-IV), Using Antibodies: A Laboratory Manual, Cells: ALaboratory Manual, PCR Primer: A Laboratory Manual, and MolecularCloning: A Laboratory Manual (all from Cold Spring Harbor LaboratoryPress), Stryer, L. (1995) Biochemistry (4th Ed.) Freeman, New York,Gait, “Oligonucleotide Synthesis: A Practical Approach” 1984, IRL Press,London, Nelson and Cox (2000), Lehninger, Principles of Biochemistry3^(rd) Ed., W. H. Freeman Pub., New York, N.Y. and Berg et al. (2002)Biochemistry, 5^(th) Ed., W. H. Freeman Pub., New York, N.Y., all ofwhich are herein incorporated in their entirety by reference for allpurposes.

Note that as used herein and in the appended claims, the singular forms“a,” “an,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a polymerase”refers to one agent or mixtures of such agents, and reference to “themethod” includes reference to equivalent steps and methods known tothose skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. All publications mentionedherein are incorporated herein by reference for the purpose ofdescribing and disclosing devices, compositions, formulations andmethodologies which are described in the publication and which might beused in connection with the presently described invention.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges is also encompassed within the invention, subject to anyspecifically excluded limit in the stated range. Where the stated rangeincludes one or both of the limits, ranges excluding either both ofthose included limits are also included in the invention.

In the following description, numerous specific details are set forth toprovide a more thorough understanding of the present invention. However,it will be apparent to one of skill in the art that the presentinvention may be practiced without one or more of these specificdetails. In other instances, well-known features and procedures wellknown to those skilled in the art have not been described in order toavoid obscuring the invention.

As used herein, the term “comprising” is intended to mean that thecompositions and methods include the recited elements, but not excludingothers. “Consisting essentially of” when used to define compositions andmethods, shall mean excluding other elements of any essentialsignificance to the composition or method. “Consisting of” shall meanexcluding more than trace elements of other ingredients for claimedcompositions and substantial method steps. Embodiments defined by eachof these transition terms are within the scope of this invention.Accordingly, it is intended that the methods and compositions caninclude additional steps and components (comprising) or alternativelyincluding steps and compositions of no significance (consistingessentially of) or alternatively, intending only the stated method stepsor compositions (consisting of).

All numerical designations, e.g., pH, temperature, time, concentration,and molecular weight, including ranges, are approximations which arevaried (+) or (−) by increments of 0.1. It is to be understood, althoughnot always explicitly stated that all numerical designations arepreceded by the term “about”. The term “about” also includes the exactvalue “X” in addition to minor increments of “X” such as “X+0.1” or“X−0.1.” It also is to be understood, although not always explicitlystated, that the reagents described herein are merely exemplary and thatequivalents of such are known in the art.

By “nucleic acid” or “oligonucleotide” or grammatical equivalents hereinmeans at least two nucleotides covalently linked together. A nucleicacid of the present invention will generally contain phosphodiesterbonds, although in some cases, nucleic acid analogs are included thatmay have alternate backbones, comprising, for example, phosphoramide,phosphorothioate, phosphorodithioate, and peptide nucleic acid backbonesand linkages. Other analog nucleic acids include those with positivebackbones; non-ionic backbones, and non-ribose backbones, includingthose described in U.S. Pat. Nos. 5,235,033 and 5,034,506. The templatenucleic acid may also have other modifications, such as the inclusion ofheteroatoms, the attachment of labels, such as dyes, or substitutionwith functional groups which will still allow for base pairing and forrecognition by the enzyme.

As used herein, a “substantially identical” nucleic acid is one that hasat least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to areference nucleic acid sequence. The length of comparison is preferablythe full length of the nucleic acid, but is generally at least 20nucleotides, 30 nucleotides, 40 nucleotides, 50 nucleotides, 75nucleotides, 100 nucleotides, 125 nucleotides, or more.

I. Methods for Generating Asymmetric Target Nucleic Acids

As summarized above, the present disclosure is generally directed toimproved methods for generating asymmetrically-tagged nucleicacids/nucleic acid libraries that find use in a number of differentdownstream applications. For example, asymmetrically-tagged nucleicacids find use as templates for carrying out nucleic acid sequenceanalysis, including single molecule sequencing analyses (e.g., SingleMolecule, Real-Time [SMRT®] Sequencing from Pacific Biosciences andnanopore-based sequencing).

In describing aspects of the methods disclosed herein, reference will bemade to the Figures. It is to be understood that the Figures merelyillustrate specific embodiments of the disclosed methods and are notintended to be limiting.

The general method typically begins with double-stranded nucleic acidfragments having defined ends, which could be blunt ends or ends withknown overhang sequences (5′ or 3′ overhangs). These nucleic acidfragments can be of any size or size range and can include DNA, RNA,DNA-RNA hybrids (e.g., molecules produced by first-strand synthesisduring cDNA preparation have one mRNA strand and one complementary DNAstrand), genomic DNA, cDNA, mRNA, tRNA, etc. In some embodiments, thenucleotide sequence of the fragments is not known.

In certain embodiments, the double-stranded nucleic acid fragments usedin methods and compositions of the present disclosure comprise nucleicacids obtained from a sample. The sample may comprise any number ofthings, including, but not limited to: bodily fluids (including, but notlimited to, blood, urine, serum, lymph, saliva, anal and vaginalsecretions, perspiration and semen) and cells of virtually any organism(e.g., mammalian species including humans); environmental samples(including, but not limited to, air, agricultural, water and soilsamples); biological warfare agent samples; research samples; theproducts of an amplification reaction (including both target and signalamplification, such as PCR amplification reactions); purified samples(e.g., such as purified genomic DNA, raw samples (bacteria, virus,genomic DNA, etc.)). As will be appreciated by those in the art,virtually any experimental manipulation may have been done on thesamples.

Genomic DNA, when used in the disclosed methods, can be prepared fromany source by three steps: cell lysis, deproteinization and recovery ofDNA. These steps are adapted to the demands of the application, therequested yield, purity and molecular weight of the DNA, and the amountand history of the source. Further details regarding the isolation ofgenomic DNA can be found in Berger and Kimmel, Guide to MolecularCloning Techniques, Methods in Enzymology volume 152 Academic Press,Inc., San Diego, Calif. (Berger); Sambrook et al., Molecular Cloning—ALaboratory Manual (3rd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory,Cold Spring Harbor, New York, 2008 (“Sambrook”); Current Protocols inMolecular Biology, F. M. Ausubel et al., eds., Current Protocols, ajoint venture between Greene Publishing Associates, Inc. and John Wiley& Sons, Inc (“Ausubel”); Kaufman et al. (2003) Handbook of Molecular andCellular Methods in Biology and Medicine Second Edition Ceske (ed) CRCPress (Kaufman); and The Nucleic Acid Protocols Handbook Ralph Rapley(ed) (2000) Cold Spring Harbor, Humana Press Inc (Rapley). In addition,many kits are commercially available for the purification of genomic DNAfrom cells, including Wizard™ Genomic DNA Purification Kit, availablefrom Promega; Aqua Pure™ Genomic DNA Isolation Kit, available fromBioRad; Easy-DNA™ Kit, available from Invitrogen; and DnEasy™ TissueKit, which is available from Qiagen. Alternatively, or additionally,target nucleic acid segments may be obtained through targeted captureprotocols where target nucleic acids are obtained initially as singlestranded segments on microarrays or other capture techniques, followedby amplification of the captured material to generate double strandedsample materials. A variety of such capture protocols have beendescribed in, e.g., Hodges E, et al. Nat. Genet. 2007 Nov. 4, Olson M.,Nature Methods 2007 November;4(11):891-2, Albert T J, et al. NatureMethods 2007 November;4(11):903-5, and Okou D T, et al. Nature Methods2007 November;4(11):907-9.

Nucleic acids that can be used in the methods described herein can alsobe derived from a cDNA, e.g. cDNAs prepared from mRNA obtained from,e.g., a eukaryotic subject or a specific tissue derived from aeukaryotic subject. Data obtained from sequencing the nucleic acidtemplates derived from a cDNA library, e.g., using a high-throughputsequencing system, can be useful in identifying, e.g., novel splicevariants of a gene of interest or in comparing the differentialexpression of, e.g., splice isoforms of a gene of interest, e.g.,between different tissue types, between different treatments to the sametissue type or between different developmental stages of the same tissuetype.

mRNA can typically be isolated from almost any source using protocolsand methods described in, e.g., Sambrook and Ausubel. The yield andquality of the isolated mRNA can depend on, e.g., how a tissue is storedprior to RNA extraction, the means by which the tissue is disruptedduring RNA extraction, or on the type of tissue from which the RNA isextracted. RNA isolation protocols can be optimized accordingly. ManymRNA isolation kits are commercially available, e.g., the mRNA-ONLY™Prokaryotic mRNA Isolation Kit and the mRNA-ONLY™ Eukaryotic mRNAIsolation Kit (Epicentre Biotechnologies), the FastTrack 2.0 mRNAIsolation Kit (Invitrogen), and the Easy-mRNA Kit (BioChain). Inaddition, mRNA from various sources, e.g., bovine, mouse, and human, andtissues, e.g. brain, blood, and heart, is commercially available from,e.g., BioChain (Hayward, Calif.), Ambion (Austin, Tex.), and Clontech(Mountainview, Calif.).

Once the purified mRNA is recovered, reverse transcriptase is used togenerate cDNAs from the mRNA templates. Methods and protocols for theproduction of cDNA from mRNAs, e.g., harvested from prokaryotes as wellas eukaryotes, are elaborated in cDNA Library Protocols, I. G. Cowell,et al., eds., Humana Press, New Jersey, 1997, Sambrook and Ausubel. Inaddition, many kits are commercially available for the preparation ofcDNA, including the Cells-to-cDNA™ II Kit (Ambion), the RETROscript™ Kit(Ambion), the CloneMiner™ cDNA Library Construction Kit (Invitrogen),and the Universal RiboClone® cDNA Synthesis System (Promega). Manycompanies, e.g., Agencourt Bioscience and Clontech, offer cDNA synthesisservices.

In some embodiments of the invention described herein, nucleic acidfragments are generated from a genomic DNA or a cDNA. There exist aplethora of ways of generating nucleic acid fragments from a genomicDNA, a cDNA, or a DNA concatamer. These include, but are not limited to,mechanical methods, such as sonication, mechanical shearing,nebulization, hydroshearing, and the like; chemical methods, such astreatment with hydroxyl radicals, Cu(II):thiol combinations, diazoniumsalts, and the like; enzymatic methods, such as exonuclease digestion,restriction endonuclease digestion, and the like; and electrochemicalcleavage. These methods are further explicated in Sambrook and Ausubel.

In further embodiments, nucleic acid molecules are obtained from asample and fragmented for use in methods of the present disclosure. Thefragments may further be modified in accordance with any methods knownin the art and described herein. Nucleic acid fragments may be generatedby fragmenting source nucleic acids, such as genomic DNA, using anymethod known in the art. In one embodiment, shear forces during lysisand extraction of genomic DNA generate fragments in a desired range.Also encompassed by the invention are methods of fragmentation utilizingrestriction endonucleases.

Double-stranded nucleic acid fragments can be any length that is desiredfor subsequent uses, e.g., cloning, sequencing, transformation,enrichment, etc. In certain embodiments, the fragments can be from about10 to about 50,000 base pairs (bp) in length and any rangertherebetween, e.g., from about 100 to about 40,000 bp, from about 300 to30,000 bp, from about 500 to 20,000 bp, from about 800 to 10,000 bp,from about 1,000 to 8,000 bp, etc. In certain embodiments, the averagesize of the double-stranded nucleic acid fragments is at least about 100bp in length, at least about 200, at least about 300, at least about500, at least about 1,000, at least about 1,500, at least about 2,000,at least about 5,000, at least about 10,000, at least about 20,000, etc.

In certain embodiments, the fragments are treated to produce blunt endsthat are compatible with ligation to a first adapter having a compatibleblunt end. Any convenient method for producing blunt ends may beemployed, including treatment with one or more enzyme having 5′ and/or3′ single strand exonuclease activity (e.g., E. coli Exonuclease III)and/or performing a fill-in reaction to extend 3′ recessed ends (e.g.,with T4 DNA polymerase). No limitation in this regard is intended.

The double-stranded fragments employed in the disclosed methods in somecases do not include a phosphate group on the 5′ hydroxyl group (5′ OH).Thus, in certain embodiments, the fragments are subjected to adephosphorylation reaction to remove the phosphate groups from the5′-terminus of both strands. In FIG. 1, the dephosphorylated 5′ ends ofdouble-stranded fragment 100 are labeled as “5′ de-P” (the 3′ ends arealso indicated for clarity). While fragment 100 is shown as having bluntends, fragments having ends with known overhang regions may also be used(e.g., “sticky” ends made by restriction endonucleases). In addition,while only one fragment is shown, this method can be used with apopulation of fragments, e.g., fragmented genomic DNA (as noted above).

In steps 1 and 2 (indicated with arrows), first hairpin adapters 102 arecontacted with and ligated to each end of dephosphorylateddouble-stranded fragment 100. However, the ligation only occurs betweenthe 3′ terminus of each strand of the double-stranded fragment and thephosphorylated 5′ end of each first adapter because of the missingphosphate groups at the 5′ termini of the fragment (the “5′ de-P”termini of fragment 100). This produces first ligation products thathave a double-stranded “insert” (corresponding to double-strandedfragment 100) and a first hairpin adapter at each end of the insert thatis ligated to the insert on only one strand. As such, each constructcomprises two nicks 104.

While steps 1 and 2 in FIG. 1 rely on dephosphorylation of thedouble-stranded fragments to produce a nick in the first ligationproducts, it is noted here that any method for producing a nick, orother site for initiation of nucleic acid synthesis, may be used. Forexample, a ligation product with no nicks at the ligation site (i.e.,where ligation of compatible ends is complete) can be treated with oneor more enzymes that create one or more single strand nick. As anotherexample, the hairpin adapter and the double-stranded nucleic acidfragments can have ends that are not fully overlapping, thereby leavinga spacer region upon ligation, which will be functionally equivalent toa nick (i.e., it can serve as a site for polymerase binding and nucleicacid synthesis, as described below). It is further noted that thenick/spacer region need not be precisely at the ligation site for thesubsequent steps of the method to produce an asymmetrically taggednucleic acid fragment. No limitation with respect to generatingnicks/spacer regions or their location in the first ligation product isintended. As such, any method for obtaining or generatinghairpin-containing fragments of the general structure of first ligationproduct 104 or a similar structure with at least one nick/spacer regionmay be used.

In steps 3 and 4, these first ligation products are contacted with astrand displacing polymerase which binds to the first ligation productsat the nick sites 104. Any convenient strand displacing polymerase maybe employed, including but not limited to a Φ29 DNA polymerase (asdescribed below). When placed under nucleic acid synthesis conditions,the polymerase begins nucleic acid synthesis from the 3′ OH group of thefirst hairpin adapter using the opposite strand of the insert as atemplate (i.e., the strand ligated to the first adapter from whichpolymerization is initiated) while simultaneously displacing thecomplementary strand of the insert in a 5′ to 3′ direction (the arrowsin FIG. 1 indicate the direction of nucleic acid synthesis; the processof strand displacement is not shown in detail). It is noted here that incertain embodiments, the first ligation product and the polymerase areincubated together under conditions that promote binding of thepolymerase to the nick site but that do not promote nucleic acidsynthesis. For example, the polymerase and the first ligation productcan be incubated in the absence of deoxyribonucleotide triphosphates(dNTPs) and/or other buffer components required for nucleic acidsynthesis (polymerization) by the polymerase.

As the polymerases proceed along the insert sequence in oppositedirections (and on different strands of the insert), the two strands ofthe insert are separated, finally resulting in two nucleic acidsynthesis products 106 (note that only one nucleic acid synthesisproduct 106 is shown in FIG. 1). Each of the nucleic acid synthesisproducts includes a fully ligated hairpin adapter at one end (i.e.,there are no nicks between the fragment and the adapter) and either ablunt or overhang terminus at the opposite end (only a blunt end isshown).

As is indicated in FIG. 1, there can be two polymerases acting on asingle first ligation product simultaneously: one initiating at eachnick (or polymerization initiation site) on opposite ends of the firstligation product. However, initiation of nucleic acid synthesis on asingle first ligation product may not be simultaneous. Thus, a firstpolymerase may initiate at a first nick in a first ligation product andpartially or fully separate the complementary strands of the insertbefore a second polymerase initiates at the second nick in the samefirst ligation product. Regardless, the resulting asymmetric molecules(i.e., the nucleic acid synthesis products) each comprise one strandfrom the original double-stranded fragment and a second strand that wassynthesized by the polymerase enzyme. It is further noted that in someinstances, nucleic acid synthesis is only initiated from one nick in afirst ligation product, and as such only a single nucleic acid synthesisproduct 106 is produced from a single first ligation product, and nottwo as indicated above.

In one aspect, the polymerase employed in this step of the methods andcompositions described herein is a Φ29 DNA polymerase or a modifiedversion thereof. Where modified recombinant Φ29 DNA polymerase isemployed, it can be homologous to a wild-type or exonuclease deficientΦ29 DNA polymerase, e.g., as described in U.S. Pat. Nos. 5,001,050,5,198,543, or 5,576,204, the full disclosures of which are incorporatedherein by reference in their entirety for all purposes. Alternately, themodified recombinant DNA polymerase can be homologous to other Φ29-typeDNA polymerases, such as B103, GA-1, PZA, Φ15, BS32, M2Y, Nf, G1, Cp-1,PRD1, PZE, SF5, Cp-5, Cp-7, PR4, PR5, PR722, L17, Φ21, or the like. Fornomenclature, see also, Meijer et al. (2001) “Φ29 Family of Phages”Microbiology and Molecular Biology Reviews, 65(2):261-287. Suitablepolymerases are described, for example, in U.S. patent applications Ser.No. 12/924701, filed Sep. 30, 2010; and Ser. No. 12/384,112, filed Mar.30, 2009. Other strand displacing polymerases can abs be used (see,e.g., International Patent Application Nos. WO 2007/075987, WO2007/075873, WO 2007/076057, incorporated herein by reference in theirentirety for all purposes).

In alternative embodiments, mechanisms other than strand displacementmay be employed to affect strand separation prior to or during nucleicacid synthesis. For example, elevation of the temperature of thereaction mixture may be used to melt the double stranded portion of thetemplate, and permit primer extension through that region. As will beappreciated, for such applications, it may be desirable to employthermally stable polymerase enzymes that are better suited to thetemperatures required for melting, and continued synthesis. A widevariety of thermostable polymerases are known in the art and areapplicable to this type of application, including, for example Taqpolymerase and its variants.

In the nucleic acid synthesis product 106 in FIG. 1, the end oppositethe adapter ligated end (i.e., lacking the first adapter) is expected tobe blunt if the polymerase traversed the entire insert, while it willhave a single-stranded 5′ overhang if nucleic acid synthesis terminatedbefore the polymerase reached the end of the insert. Further, if thepolymerase employed has terminal transferase activity, there may be a 3′overhang instead of a blunt end if the polymerase traverses the entireinsert. No limitation in this regard is intended.

As shown in FIG. 1, the asymmetric nucleic acid synthesis product 106 istreated to create an end opposite to the first adapter that is able tobe fully ligated with a second adapter 108 (sometimes called“polishing”; see step 5 in FIG. 1). In certain embodiments, thesynthesis product 106 is treated to create blunt and 5′ phosphorylatedends. Thus, in certain embodiments, synthesis product 106 is treatedwith one or more enzymes having 5′ and/or 3′ single stranded exonucleaseactivity (e.g., E. coli Exonuclease VII), one or more enzymes forfilling in 3′ recessed ends (e.t., T4 DNA polymerase), and/or one ormore enzymes for adding a phosphate to the 5′ OH group (see 110 in FIG.1). This treatment is done under appropriate reaction conditions suchthat the enzymes display their intended activities (conditions known inthe art). The non-adapter ligated end of the resulting treated synthesisproduct has had any 3′ or 5′ single-stranded overhangs removed/repairedand has a phosphate at the 5′ end (110). In some cases, the singlehairpin product 106 produced in step 4 is used directly or is furthermodified and used as a single hairpin single molecule sequencingtemplate. In some cases it is desired to add a second hairpin to theproducts 106 to produce asymmetric circular constructs as illustrated insteps 5 and 6.

In steps 5 and 6 in FIG. 1, a second hairpin adapter 108 having at leastone nucleotide sequence difference than the first hairpin adapter (andwhich has a compatible blunt end) is contacted with and ligated to theblunt end of the blunt end-polished nucleic acid synthesis product togenerate a second ligation product having a different hairpin adapter ateach end 112. It is noted that because the 5′ end of both the secondadapter and the blunt-end synthesis product were phosphorylated, thesecond ligation product does not have a nick (as did the first ligationproduct. Second ligation product 112 is thus an asymmetric nucleic acidconstruct having different hairpin adapters at each end, which issometimes referred to as a “closed asymmetric molecule” (meaning thatthe construct comprises one contiguous nucleic acid strand having twocomplementary strands that can for a double-stranded region and twoadapters connecting the complementary strands at each end).

FIG. 1 provides an illustration of one embodiments of the presentdisclosure in which a fully 5′ dephosphorylated fragment is processed togenerate asymmetrically-tagged product 112. However, small deviations inthe process can generate product having alternative, but still veryuseful, structures. Two such deviations are shown in FIGS. 2 and 3.

FIG. 2 starts with a first ligation product in which only one end hasbeen ligated with a first hairpin adapter, and thus includes only 1 nick104. When this first ligation product is contacted with a stranddisplacing polymerase and placed under nucleic acid synthesis conditions(steps 3 and 4; as described above), the polymerase displaces strand200. However, because strand 200 does not have an adapter ligated to its3′ end, it cannot serve as a template for nucleic acid synthesis.Consequently, in subsequent steps strand 200 will be removed from thereaction (e.g., by exonuclease treatment). However, the resultingsynthesis product 106 from steps 3 and 4 is identical in structure tothose produced in FIG. 1 and thus can be processed in subsequent stepsto ligate the second adapter.

FIG. 3 starts with a fragment 300 in which one 5′ terminus has not beende-phosphorylated 302 (labeled “5′ P”) and thus when the first adapteris ligated to this fragment, the resulting first ligation productcontains only one nick 104 (on the dephosphorylated 5′ end of thefragment). When this first ligation product is contacted with a stranddisplacing polymerase and placed under nucleic acid synthesisconditions, the polymerase uses the first strand of the insert, theadapter at the opposite end, and the complementary strand of the insertas a template to produce the nucleic acid synthesis product 306 (steps 3and 4; arrow 304 indicates newly synthesized strand). Nucleic acidsynthesis product 306 this includes two regions with nucleic acidsequences derived from the insert (308 and 310) and a region 312 derivedfrom the adapter. While synthesis product 306 is not identical instructure to those produced in FIG. 1, it can still be processed insubsequent steps to ligate the second adapter.

The methods disclosed herein provide an improvement over current methodsin which two different adapters are ligated simultaneously todouble-stranded fragments having blunt ends. Specifically, the disclosedmethods can produce a very high percentage (approaching 100%) ofasymmetrically-tagged target nucleic acids whereas methods in which bothadapters are simultaneously ligated to blunt-ended fragments, only about50% of the resulting tagged molecules will be asymmetric (50% will havethe same adapter at both ends, whether it be the first or secondadapter). Further, because there is no requirement that the sequence ofthe double-stranded fragments be known, the methods can be used forgenerating libraries of fragments having unknown sequence, e.g., for denovo sequencing applications.

While the methods disclosed herein do provide a higher percentage ofasymmetrically-tagged nucleic acids, there are methods for starting witha mixed population of adapter-tagged nucleic acids (containingasymmetrically- and symmetrically-tagged nucleic acids) and enrichingfor the desired species (asymmetrically-tagged nucleic acid). Forexample, asymmetrically-tagged nucleic acid fragments can be isolatedfrom a mixed population using one or more binding reagents that arespecific for one of the adapters, e.g., a binding reagent that binds toa first adapter but not to a second adapter (e.g., an oligonucleotidethat specifically hybridizes to a single stranded region in the firstadapter that is not present in the second adapter). Any desired bindingreagent can be employed, e.g., oligonucleotides, members of bindingpairs (e.g., biotin/avidin, antigen/antibody, etc.), and the like.

As but one example, a mixed population containing symmetrically-taggednucleic acid fragments having adapter A at each end andasymmetrically-tagged nucleic acid fragments having hairpin adapter A atthe first end and hairpin adapter B at the second end can be contactedwith a biotin labeled oligonucleotide specific for a single strandedregion in hairpin adapter B under nucleic acid hybridization conditions.After hybridization, the fragments containing hairpin adapter B can beenrich by contacting the oligonucleotide-hybridized sample toimmobilized streptavidin moieties (e.g., streptavidin coated beads)under appropriate binding conditons and washing away the non-bindingspecies. The bound fragments can be eluted in any convenient manner,e.g., chemical or heat denaturation to melt the oligonucleotide from itscognate binding site in hairpin adapter B. After elution, additionalnucleic acid fragment clean-up steps can be performed.

It is noted here that multiple adapter-specific isolation steps may beperformed to enrich for asymmetrically-tagged nucleic acid fragments,e.g., a first round for isolation of fragments containing the firstadapter (adpter A) followed by a second round for isolation of fragmentscontaining the second adapter (adapter B) from the fragments isolated inthe first round. Any fragments enriched by this process would thus haveto have both adapter A and adapter B (i.e., be asymmetrically tagged).

The asymmetrically-tagged target nucleic acids generated according tothe methods disclosed herein find use in a number of differentdownstream applications, which in general can be determined by the user.

One benefit to the closed asymmetric molecule is that undesired nucleicacids that do not have hairpin adapter sequences at both ends can beremoved from the preparation with exonuclease treatment. Further, theseconstructs are ideal for redundant or iterative sequencing in asequencing reaction that can process circular template molecules, sincethe molecule is essentially a single-stranded circle with internalcomplementarity within the insert portion. An example of a sequencingtechnology that can be employed to sequence these closed asymmetricmolecules is single molecule, real-time (SMRT®) Sequencing from PacificBiosciences. When used in a SMRT® Sequencing reaction, a closedasymmetric molecule may be referred to as an asymmetric SMRTBELL®template.

It is noted that while the second adapter 108 in FIG. 1 is a hairpinadapter, in certain embodiments, the second adapter is not a hairpinadapter. Examples of different adapters that find use in aspects of thepresent disclosure are shown in FIG. 4. In this figure, blunt-end andphosphorylated nucleic acid synthesis product 400 can be ligated tobubble adapter 402, fully double-stranded linear adapter 404, overhangadapter 406 (note that only 3′ overhang is shown but 5′ overhang is alsopossible), or Y adapter 408 to produce any of the second ligationproducts shown at the right 410. In addition, these second adapters caninclude any desired functional sequences or moieties, including primerbinding sites, barcodes, registration sequences, capture moieties ornucleic acid sequences (e.g., biotin moieties, poly-A sequences, etc.),and the like. For example, an overhang adapter (e.g., adapter 406 inFIG. 4) can include a poly-A capture sequence in the single strandedregion that enables capture on poly-T-coated paramagnetic beads (orother solid surface). No limitation in this regard is intended. Such“partially closed asymmetric molecules” find use, for example, insequencing methods that utilize linear, single-stranded templates, suchas nanopore sequencing, Illumina sequencing, Sanger sequencing, IonTorrent sequencing, pyrosequencing, and others widely known in the art.

Thus, in some embodiments, the asymmetrically-tagged target nucleicacids produced according to the disclosed methods, whether closed orpartially closed, are used directly in sequencing reactions. Further,the asymmetrically-tagged target nucleic acids produced according to thedisclosed methods may be subjected to a size selection prior to use indownstream assays/analyses.

Although creating nicked symmetric molecules is one preferred strategyto provide initiation sites for polymerase-mediated synthesis andstrand-displacement, other strategies can also be used. For example, anadapter with an extended stem region containing a small number of RNAbases could be used in the initial ligation. Subsequent digestion withan RNase (e.g., RNaseH2) would remove the RNA bases, leaving a free 3′OH and a short gap for polymerase binding and initiation of nucleic acidsynthesis.

It is further noted that in certain embodiments, modified nucleotidesare used when performing the nucleic acid synthesis step such to allowthe original nucleic acid strand and the synthesized (or nascent) strandin the nucleic acid synthesis product to be distinguished from oneanother, e.g., during sequencing.

The asymmetrically-tagged target nucleic acids of the present disclosure(also referred to as “templates” when used for sequence determination)provide numerous advantages over simple linear template sequences, andeven other circular template sequences (see, e.g., U.S. Pat. No.7,302,146 for a discussion of circular templates for sequencingapplications, the full disclosure of which is incorporated herein byreference in its entirety for all purposes). In particular, as withcircular templates, the template configurations of the presentdisclosure allow for single molecular consensus sequence determination,where sequencing a given template provides duplicative or replicate dataof the sequence information obtained, and thereby improves accuracy overlinear templates by providing multiple reads for a given templatesequence or sequence portion, that can be used to derive consensussequence data from a given template sequence and/or for specific baselocations within such sequence. In these templates, the potential forconsensus sequence determination is provided, in one respect, by virtueof the circular nature of the overall template structure, for acompletely contiguous template, allowing repeated processing of the samemolecule to obtain consensus base calls and/or a consensus sequence. Inaddition or alternatively, the templates of the present disclosure, byvirtue of their inclusion of double stranded segments, provide consensussequence determination through the sequencing of both the sense andantisense strand of such sequences (in both the partially and completelycontiguous configurations).

The ability to asymmetrically tag nucleic acid fragments provides amechanism to incorporate different functionalities at each end of anucleic acid molecule. Examples of elements or regions that provide suchfunctionalities include, but are not limited to: primer binding sites,restriction enzyme recognition sites, modified bases, enrichmentfunctionalities, barcode sequences, and the like. Thus, the disclosedmethods provide a convenient way to generate asymmetrically barcodednucleic acid libraries, e.g., genomic or cDNA libraries. Such librariescan be used, e.g., in multiplexed genetic analyses.

For example, in some cases, adapter sequences may be included asregistration sequences to provide landmarks within the overall templatesequence, e.g., to provide alignment of iterative sequence data, toidentify the level of coverage in a consensus sequence read, to identifypoints in a sequencing process where one is progressing into a consensussequence, e.g., an antisense strand or repeated sequence of the entiretemplate, and the like.

In addition, such sequences may provide control opportunities for thesequencing process using such templates. For example, one mayincorporate primer recognition sequences within an adapter to initiatepolymerization. As noted previously, the flexibility as to the types andconfiguration of the primer sequences is increased by virtue of immunityfrom binding to the target portion of the sequence, which exists as adouble stranded segment.

Additional control sequences may also be provided, e.g., sequences thatallow control over the initiation of synthesis, e.g., through ahybridized probe or reversibly modified nucleotide, or the like (see,e.g., U.S. Patent Application No. 20080009007, the full disclosure ofwhich is incorporated herein by reference in its entirety for allpurposes.). Other control sequences may include binding sites fortranscription factors. For example, repressor binding regions may beprovided as control sequences within an adapter, such as the lacrepressor recognition sequence, which when bound by the lac repressorprotein, has been shown to block replication both in vivo and in vitro.Reinitiation of replication is accomplished through the addition ofappropriate initiators, such as isophenylthiogalactoside (IPTG) orallolactose. Other DNA binding protein recognition sites may also beincluded within an adapter to allow control over the progress ofsynthesis using the templates of the invention. Other controllableelements may include the use of non-natural bases (also termed 5^(th)bases) within an adapter which are not paired with any of the four basicnucleoside polyphosphates in the synthesis reaction. Upon encounteringsuch a base, the polymerase would pause until its own particularcomplement was added to the reaction mixture. Likewise, an engineeredpause point within an adapter could include a “damaged” base that causesa stop in replication until repair enzymes are added to the mixture. Forexample, a base position having a pyrimidine dimer could be includedwithin an adapter. Such compounds would cause the replication complex topause. Addition of the photolyase DNA repair enzyme would repair theproblem location and allow replication, and sequencing to continue.

Recognition sites for a variety of other oligonucleotide probes can alsobe incorporated into adapter sequences, e.g., hybridization sites forlabeled probes, molecular beacons, TAQMAN® probes (Applied Biosystems),INVADER® probes (Third Wave Technologies, Inc.), or the like, that canbe used to provide other indications of the commencement of synthesis.Additionally, non-native bases that interact/complement other non-nativebases may be used to provide an initiation point for synthesis andsequencing.

In some cases, it may be desirable to provide endonuclease recognitionsites within an adapter, which can allow for a mechanism to release agiven template sequence from a synthesis reaction, i.e., by linearizingit, and allowing the polymerase to run off the linear template, and/orto expose the template to exonuclease activity, and thus terminatesynthesis through removal of the template. Such sites could additionallybe exploited as control sequences by providing specific bindinglocations for endonucleases engineered to lack cleavage activity, butretain sequence specific binding.

The asymmetrically-tagged nucleic acid templates described hereinfurther find use in methods for loading single molecule compositionsinto array regions as described in U.S. patent application Ser. No.15/078,915, entitled “METHODS AND COMPOSITIONS FOR SINGLE MOLECULECOMPOSITION LOADING”, filed on Mar. 23, 2016 the entirety of which ishereby incorporated by reference herein.

In addition, asymmetrically-tagged nucleic acid templates are expectedto have improved loading characteristics in ZMW (or othernanometer-scale well structures) as compared to symmetrically-taggedtemplates due to steric considerations. For example, because of thepresence of identical adapters on the ends, symmetrically taggedtemplates can have two polymerases bound, leading to less efficientloading. In addition, if such complexes do load, having two polymerasesin a single ZMW leads to shorter reads and/or noisy data, as bothpolymerases will be working on the same substrate at the same time.

II. Kits and Systems

The present disclosure also provides applied embodiments of the methodsand compositions disclosed herein.

For example, in certain embodiments, the present disclosure provideskits that are used in preparation of the asymmetrically-tagged nucleicacid constructs described herein. A first exemplary kit provides thematerials and methods for preparation of the asymmetrically-taggednucleic acid constructs in accordance with the invention, as describedelsewhere herein. As such, the kit will typically include thosematerials that are required to prepare asymmetrically-tagged nucleicacid constructs as outlined herein, e.g., in accordance with the variouspreparation processes outlined above. As will be appreciated, dependingupon the nature of the asymmetrically-tagged nucleic acid construct andthe method used, the kit contents can vary. For example, where one isemploying different hairpin adapters (first and second adapters) thatare to be coupled to opposite ends of double stranded nucleic acidsegments, the kits will typically include such different hairpinadapters (e.g., with blunt ends having 5′ phosphate groups), along withappropriate ligation enzymes and protocols for attaching such adaptersto the opposite ends of double stranded nucleic acids, as well as anyprocessing enzymes that may be desirable for treating the ends of thedouble stranded segments prior to ligation, e.g., phosphatases,exonucleases, and the like to provide blunt end nucleic acids lacking 5′phosphate groups. In some cases, these kits may include enzyme systemsfor providing 5′ phosphate groups to the ends of fragments (e.g., firstligation products as described above). The kits may further includereagents for performing nucleic acid synthesis reactions, including butnot limited to a strand displacing polymerase and buffers/regents forbinding the polymerase to a nick site in a nucleic acid fragments aswell as for initiating and supporting nucleic acid synthesis from thenick site. As the polymerase binding and nucleic acid synthesis stepsmay be performed under different reaction conditions, separatebuffers/reagents can be provided for each. Alternatively, a single setof buffers/reagents for simultaneous polymerase binding and nucleic acidsynthesis may be provided.

In addition, kits may include reagents for removing undesired nucleicacids in the sample during or after adapter ligation, includingexonucleases, nucleic acid purification columns or beads, size-selectioncolumns or spin tubes, affinity/capture reagents (e.g., biotin, avidin,capture primers, etc.). Further, kits may include reagents forgenerating the initial nucleic acid fragments to be tagged, includingnucleic acid isolation reagents, fragmentation reagents (e.g.,fragmentation columns, restriction enzymes, etc.).

A second exemplary kit provides materials and methods not just for thepreparation of the asymmetrically-tagged nucleic acid constructs of theinvention, but also for the use of such asymmetrically-tagged nucleicacids in performing sequence analysis on target nucleic acid sequences(when used in sequence analyses, the asymmetrically-tagged nucleic acidconstructs are sometimes referred to as “templates”). Thus, in additionto the materials and methods set forth above, such kits may additionallyinclude reagents used in such sequencing processes, such as primersequences for initiating the sequence process, polymerase enzymes, andsubstrates that provide for optical confinement of nucleic acidsynthesis complexes. In certain aspects, such substrates will typicallyinclude one or more arrays of zero mode waveguides (ZMW). Such waveguidearrays may further include surface treatments that provide for enhancedlocalization of synthesis complexes within the illumination volumes ofsuch zero mode waveguides, e.g., as described in Published InternationalPatent Application No. WO 2007/123763, incorporated herein by referencein its entirety for all purposes. Additionally, such kits may optionallyinclude nucleotide compositions for use in sequencing applications,including, for example labeled nucleotides that include fluorescent orotherwise detectable labeling groups coupled to the phosphate groups ina nucleoside polyphosphate construct at a phosphate group other than thealpha phosphate. A variety of other types of labeled and unlabelednucleotides may be optionally includes within the kits and are generallyknown in the art.

III. Protocol

The protocol described below provides one embodiment for producingasymmetrically-tagged nucleic acids and is not intended to be limiting.

1. Shear gDNA to generate a sample containing fragments having a desiredaverage DNA length. This can range from 100 bp to >20 kb.

2. Create blunt ends by contacting the fragments with one or more enzymehaving 5′ and 3′ single strand exonuclease activity under appropriateconditions (e.g., E. coli Exonuclease VII).

3. Perform a DNA repair incubation to repair degradation of thefragments due to fragmentation process (e.g., using a kit sold by NewEngland Biolabs; NEB).

4. Purify the DNA fragments (e.g., using AMPure beads).

5. Dephosphorylate the 5′ ends of the fragments by treating withphosphatase (e.g., Antarctic Phosphatase; NEB).

6. Purify the DNA fragments (e.g., using AMPure beads).

7. Ligate first hairpin adapter using DNA ligase.

8. Purify the DNA fragments (e.g., using AMPure beads).

9. Bind polymerase to nick sites (e.g., Φ29 polymerase or derivative).

10. Extend by adding appropriate buffers and dNTPs and incubating atappropriate temperature.

11. Purify the DNA fragments (e.g., using AMPure beads).

12. Measure DNA concentration to QC.

13. Create blunt ends by contacting the adaptered fragments with one ormore enzyme having 5′ and 3′ single strand exonuclease activity underappropriate conditions (e.g., E. coli Exonuclease VII).

14. Perform a DNA repair incubation to repair degradation of thefragments due to fragmentation process (e.g., using a kit sold by NewEngland Biolabs; NEB).

15. Purify the DNA fragments (e.g., using AMPure beads).

16. Ligate second adapter using DNA ligase.

17. Perform exonuclease digestion to remove single stranded moleculesand non-ligated fragments/adapters (e.g., using E. coli Exonucleases IIIand VII).

18. Purify the DNA fragments (e.g., using AMPure beads).

19. Measure DNA concentration to QC.

Initial use of this protocol has yielded in the range of 1-2 μg ofasymmetrically tagged nucleic acid templates from 10 μg of startingmaterial (i.e., 10 μg of double-stranded DNA fragments).

Although described in some detail for purposes of illustration, it willbe readily appreciated that a number of variations known or appreciatedby those of skill in the art may be practiced within the scope ofpresent invention. To the extent not already expressly incorporatedherein, all published references and patent documents referred to inthis disclosure are incorporated herein by reference in their entiretyfor all purposes.

1. A method of producing an asymmetrically-tagged nucleic acid,comprising: providing a double-stranded nucleic acid fragment comprisingidentical hairpin adapters at each end; treating the double-strandednucleic acid fragment with one or more enzymes that create a singlestrand nick in the double-stranded nucleic acid fragment; contacting thedouble-stranded nucleic acid fragment to a polymerase under nucleic acidsynthesis conditions, wherein the polymerase initiates template-dependennucleic acid synthesis from the single strand nick in thedouble-stranded nucleic acid fragment to produce a nucleic acidsynthesis product; ligating a second adapter to the nucleic acidsynthesis product, wherein the second adapter is ligated to the endopposite the hairpin adapter on the nucleic acid synthesis product, andwherein the second adapter has at least one difference as compared tothe hairpin adapter, thereby producing an asymmetrically-tagged nucleicacid.
 2. The method of claim 1, wherein the providing step comprisesfragmenting a nucleic acid sample and ligating hairpin adapters to theends of the fragmented nucleic acids to generate the double-strandednucleic acid fragments.
 3. (canceled)
 4. The method of claim 1, whereinthe second adapter is a second hairpin adapter.
 5. The method of claim1, wherein the second adapter is selected from the group consisting of:a bubble adapter, double-stranded linear adapter, a linear overhangadapter, and a Y adapter.
 6. (canceled)
 7. The method of claim 1,wherein the polymerase is a strand displacing polymerase enzyme.
 8. Themethod of claim 7, wherein the strand displacing polymerase is selectedfrom the group consisting of: a Φ29 DNA polymerase or modified versionthereof, a homolog of a Φ29 DNA polymerase or modified version thereof,and combinations thereof.
 9. The method of claim 1, the method furthercomprising contacting the nucleic acid synthesis product with a kinaseunder conditions for phosphorylating the free 5′ end of the nucleic acidsynthesis product.
 10. The method of claim 1, further comprisingdetermining a nucleotide sequence of at least one strand of theasymmetrically-tagged nucleic acid using a template directed, polymerasemediated nucleic acid sequencing process.
 11. The method of claim 10,wherein the sequencing process sequences each strand of the doublestranded region of the asymmetrically-tagged nucleic acid.
 12. Themethod of claim 10, wherein the sequencing process sequences at leastone strand of the double stranded nucleic acid segment more than once.13. The method of claim 1, wherein at least one of the hairpin adapterand the second adapter comprises a primer recognition sequence.
 14. Themethod of claim 1, wherein at least one of the hairpin adapter and thesecond adapter comprises a barcode sequence. 15-21. (c